apoptosis and oncotic necrosis apoptosis vs oncotic necrosis initiation execution apoptosis...

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  • Apoptosis and Oncotic Necrosis: The final common pathways of liver injury

    Hartmut Jaeschke

    Department of Pharmacology, Toxicology & Therapeutics

    University of Kansas Medical Center Kansas City, KS

  • Fibrosis: Response to Cell Injury

    Rockey & Friedman, 2006

    Chronic Cell Injury: Apoptosis and Oncotic Necrosis

  • • cellular condensation (cell shrinkage)

    • membrane blebbing, but no loss of integrity

    • aggregation of chromatin at the nuclear membrane

    • formation of membrane bound vesicles (apoptotic bodies)

    • no disintegration of organelles; organelles remain intact

    • swelling of the cell and lysis

    • loss of membrane integrity

    • flocculation of chromatin

    • no vesicle formation, complete lysis

    • disintegration (swelling) of cell organelles

    Morphology

    Apoptosis Oncotic Necrosis

  • Hepatocellular Apoptosis

    H & E

    Galactosamine (500 mg/kg; 6 hr)

  • Hepatocellular Oncotic Necrosis and Inflammation

    H & E

    Bile Duct Ligation 3 days

  • Apoptotic Cell Death: Caspases

    Proenzyme (zymogen)

    prodomain large subunit linker domain small subunit

    Peptidase 1 496

    DED DED Pro-Caspase 8 Initiator Caspase

    Death effector domainDED

    Peptidase 1 277 Pro-Caspase 3

    Effector Caspase

    Cysteine-dependent aspartase domainPeptidase

    PeptidaseCARD 1 416 Pro-Caspase 9

    Initiator Caspase

    Caspase recruitment domainCARD

  • Activation of Caspases Proenzyme (zymogen)

    Active Caspase (tetramer)

    prodomain large subunit linker domain small subunit

  • Activation of Caspases Proenzyme (zymogen)

    Active Caspase (tetramer)

    prodomain large subunit linker domain small subunit

    0 20 40 90 120 Time (min)

    p32

    p11 Fas-Ab Jo-2

    Proenzyme

    Active Fragment

  • 0

    2 0 0

    4 0 0

    6 0 0

    Activation of Caspases

    0 20 40 90 120 Time (min)

    p32

    p11

    (ΔF/min/mg protein)

    600

    400

    200

    0

    *

    *

    *

  • DDDD DD

    FAS-L FAS-L

    DDDD DD

    FA D

    D FA

    D D

    FA D

    D FA

    D D

    Cas 8

    FAS-L

    DDDD DD

    FA D

    D FA

    D D

    FA D

    D FA

    D D

    Cas 10

    C as

    p1 0

    Casp 6

    C asp

    8Bid

    Ba xB ak

    C

    C

    C

    C

    C

    C

    C

    C C

    C

    C

    C C

    C

    Casp 7 Casp 3A

    pa f1

    A pa

    f1

    C as

    p 9

    C as

    p 9

    C

    C C

    C

    CAD

    En do

    G En

    do G

    CAD

    En do

    G

    A IF

    A IF

    D ia

    bl o

    D iablo

    C

    B ak B ak

    B ax

    B ax

    Caspase Substrates

    CAD

    CARD

    C

    C

    CARD

    DISC

    FAS-L FAS-L

    dATP

    dATP

    Fas-mediated Apoptosis Signaling in Hepatocytes

    CAD ICADCAD

  • Bid

    Bcl-2

    Ba k

    C

    C

    C C

    C

    C

    C

    C

    C

    C

    C

    C Casp 3

    C

    C

    C C

    EndoG EndoG

    EndoG

    EndoG

    AIF

    Diablo

    C

    Caspase 3 SubstratesAIF

    cIAPDiablo

    Intrinsic Pathway of Apoptosis

    Ca

    Ca

    Ca

    Ca

    AIF

    Diablo

    Diablo C

    AIF

    C

    C

    C

    C

    C

    C

    C C

    CC

    C

    C

    BaxBakBax

    Bak

    AIF Diablo

    Ca Ca

    Ca

    Ca

    Ca Ca

    Ca Ca

    Ca Ca

    PTP

    Bcl-XL

    Bcl-2 Bax

    Bax

    Bax

    28S

    eIF2α

    PT P

    Cathepsin

    C

    A pa

    f1CARD

    Apaf1CA R

    D

    Apaf1 CA RD

    A paf1

    CARDCasp 9

    Ca sp

    9C asp

    9

    C

    C C

    Bax BakBax

    Bak

    CaCa Ca

    Ca

    Casp 9

    C C

    m-Calpain

    Casp 12

    p53Bax

  • Caspase Targets in Apoptosis Casp10

    Casp 6

    Casp 8

    Casp 7

    Casp 3

    A pa

    f1

    A pa

    f1

    C as

    p 9

    C as

    p 9

    CARD C

    CARD

    CARD Casp 2

    MDM2Fodrin

    Prese- nelin2 Gelsolin

    Actin Lamin A

    DNA fragmentation and chromatin condensation

    Cytoskeleton Cell shape and membrane

    blebbing

    CAD

    Trans- glutaminase

    Topo- isomerase

    PARPDNA-PKEndoGICAD

    Keratin- 18

    Gas2

    Cell cycle and other

    p21

    FAK

    hnRNP

    β-catenin

    NuMASREB1

    Calpastatin

    EMAP II

    Rock-1

  • DNA Fragmentation

    1 2 3 4 5 6 7 8 9 10 11 12 13

    MW Controls G/ET

    0

    200

    400

    600

    800

    1000

    (% Vmax)

    0 0 20 40 90 120

    600

    400

    200

    800

    1000 *

    *

    *

    Time (min)

    DNA LadderAnti-Histone ELISA

    CADCAD

  • DNA Strand Breaks: TUNEL Assay

    Terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling

    GalactosamineGalactosamine (500 mg/kg; 6 hr)(500 mg/kg; 6 hr)

  • Characteristic Features of Oncotic Necrosis

    • Morphology: cell swelling, cell contents release, karyolysis

    Mechanism is dependent on the insult

  • Oncotic Necrosis: Acetaminophen- induced Hepatotoxicity

    Cell Swelling, Karyolysis

    Cell Contents Release : Plasma ALT > 3000 U/L)

  • DNA Strandbreaks during Oncotic Necrosis

    TUNEL Assay

    CV

    4 h APAP 6 h APAP

  • DNA Strandbreaks: Oncotic Necrosis vs Apoptosis

    TUNEL Assay

    CV

    6 h Gal/ET 6 h APAP

  • DNA Fragmentation: Apoptosis vs Oncotic Necrosis

    1 2 3 4 5 6 7 8 9 10 11 12 13

    MW Controls G/ET

    DNA LadderAnti-Histone ELISA

    APAP

    D N

    A F

    ra gm

    en ta

    tio n

    (% c

    on tro

    l)

    0

    500

    1000

    1500

    2000

    C G/E G/E ZVAD

    AAP AAP ZVAD

    AAP GSH

    *

    * * #

    #

  • 1 2 3 4 5 6 7 8 9

    0 0.5 1 2 3 4 6 G/E

    Acetaminophen (h)

    p32

    p11

    Caspase-3 Processing during Acetaminophen Toxicity

    Lawson et al., Toxicol Appl Pharmacol 156: 179-86, 1999 Gujral et al., Toxicol Sci 67: 322-8, 2002

  • Ba xB ak

    C

    C

    C

    C

    C

    C

    C

    C C

    C

    C

    C C

    C

    C

    C C

    C

    EndoG

    En do

    G En

    do G

    EndoG

    En do

    G

    EndoG

    A IF

    A IF

    D ia

    bl o

    D iablo

    C

    B ak B ak

    B ax

    B ax

    AIF

    C

    Diablo AIF

    EndoG

    DNA Fragmentation: Nuclear Translocation of Mitochondrial Intermembrane Proteins

    Diablo

    C C

    Nucleus

    Mitochondria

    Control

    5 mM AAP, 6 h

    Endonuclease G

    Bajt et al., Toxicol Sci 94: 217-225,2006

  • Cytochrome c

    Bax

    Bid tBid

    Co ntr

    ol AP

    AP 2

    h

    AP AP

    4 h

    Co ntr

    ol AP

    AP 2

    h AP

    AP 4

    h

    Mitochondria Cytosol

    APAP-induced Bax and tBid Translocation to Mitochondria

  • APAP Hepatotoxicity in Bax Gene Knockout Mice

    P la

    sm a

    A LT

    (U /L

    )

    0

    1000

    2000

    3000

    4000

    5000

    6000

    7000

    *

    WT Bax-/-

    Bajt et al., J Pharmacol Exp Therap, in press, 2007

  • APAP Hepatotoxicity in Bax Gene Knockout Mice

    P la

    sm a

    A LT

    (U /L

    )

    0

    1000

    2000

    3000

    4000

    5000

    6000

    7000

    *

    WT Bax-/-

    Bajt et al., J Pharmacol Exp Therap, in press, 2007

    WT

    Bax-/-

  • NAPQI APAP GSH ↓

    Protein Arylation

    P4501.

    2.

    Bax

    Cyt c / Smac AIF Endonuclease G

    ONOO-

    Nucleus

    DNA- Strandbreaks

    ATP ↓

    Caspase Activation

    ATP ↓

    PARP Activation

    DNA Repair

    DNA- Fragmentation

    Chromatin Condensation

    NAD+ Depletion

    ATP ↓ ⇐ MPT

    ? Mechanism of Mechanism of APAP ToxicityAPAP Toxicity

    Toxicol Sci 89: 31-41, 2006

    O2 – NO

    Bax MPT

    Protein Nitration

  • Apoptosis vs Oncotic Necrosis

    Initiation Execution

    Apoptosis Apoptosis ATP high